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Increased intracellular sequestration of the insulin-regulated aminopeptidase upon differentiation of 3T3-L1 cells.

ABSTRACT: In fat and muscle cells, the glucose transporter GLUT4 is sequestered in an intracellular compartment under basal conditions and redistributes markedly to the plasma membrane in response to insulin. Recently, we characterized a membrane aminopeptidase, designated IRAP (insulin-regulated aminopeptidase), that colocalizes with intracellular GLUT4 and similarly redistributes markedly to the plasma membrane in response to insulin in adipocytes. In contrast to GLUT4, IRAP is also expressed in 3T3-L1 fibroblasts, and this finding provided an opportunity to compare its subcellular distribution in fibroblasts and adipocytes. The relative amount of IRAP at the cell surface was measured by a cell surface biotinylation method. The portion of total IRAP at the cell surface in unstimulated adipocytes was 30% of that in unstimulated fibroblasts. Upon insulin treatment the portion of IRAP at the cell surface was the same in fibroblasts and adipocytes, and was increased 1.8-fold in fibroblasts and 8-fold in adipocytes. A similar analysis of the distribution of the transferrin receptor (TfR), the paradigm for recycling plasma membrane receptors, revealed that the portions of the TfR at the cell surface in both the basal and insulin-treated states were almost unchanged upon differentiation, and that insulin caused an increase of about 1. 6-fold in the amount of TfR at the cell surface. These results show that enhanced intracellular sequestration of IRAP occurs during adipogenesis, and that this effect underlies the larger insulin-elicited fold increase of IRAP at the cell surface in adipocytes.

SUBMITTER: Ross SA

PROVIDER: S-EPMC1219237 | biostudies-other | 1998 Mar

REPOSITORIES: biostudies-other

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Project description:BackgroundObesity and metabolic syndrome are important public concerns, and there is increasing demand for effective therapeutic strategies. Flavonoids are expected to improve the risk factors associated with metabolic syndrome. Anthocyanidins are a kind of flavonoids; well known for their anti-oxidative, anti-inflammatory and anti-tumor properties. However, their effects on adipocytes and molecular systems are not well defined. In this study, we examined the effects of anthocyanidins-enriched bilberry extracts on adipocyte differentiation.MethodsUtilizing 3T3-L1 cell line, we investigated that bilberry extracts and anthocyanidins induced inhibition of lipid accumulation during adipogenesis. To identify what is the most important bilberry mediated-effect, we analyzed the expressions of key transcriptional factors associated with adipocyte differentiation by Real Time (RT)-PCR. From the results of RT-PCR, we hypothesized that bilberry extracts and anthocyanidins blocks insulin signal, we determined the phosphorylation of tyrosine residues of insulin receptor substrate 1 (IRS1) protein by western blotting analysis. In addition, we compared the whole-genome expression profiles of early stage of adipocyte differentiation under four different growth conditions (DMSO, bilberry, two anthocyanidins) by microarray analyses and Gene Set Enrichment Analysis (GSEA).ResultsExposure to bilberry extracts and anthocyanidins during adipocyte differentiation inhibited 3T3-L1 differentiation. During this period, bilberry extracts and anthocyanidin significantly decreased a key adipocyte differentiation-associated marker, peroxisome proliferator-activated receptor- γ (Ppar γ ) and sterol regulatory element-binding protein 1c (Srebp1c). Western blotting analysis showed that bilberry extracts and anthocyanidin decreased the phosphorylation of tyrosine residues of IRS1. In addition, microarray experiments and GSEA data revealed significantly altered expression of the known genes of the insulin pathway in cells treated with bilberry extracts or anthocyanidins in the early differentiation stages.ConclusionsOur data demonstrate that anthocyanidin enriched bilberry extracts strongly inhibit the adipocyte differentiation via the insulin pathway. Furthermore, bilberry extracts might be used as a potential complementary treatment for the obese patients with metabolic syndrome.

Project description:BackgroundAdipose tissue is a critical regulator of lipid storage and endocrine function. Impairment of the recruitment of new adipocytes in the adipose tissue is associated with ectopic fat accumulation, diabetes and insulin resistance. Torreya nucifera, an evergreen conifer that grows in warm temperate climates, has been found to exert beneficial effects against inflammation, infection and diabetes. However, the molecular mechanisms responsible for these effects at the cellular level remain unknown. This study aimed to investigate effects of Torreya nucifera seed oil (TNSO) on 3T3-L1 adipocyte differentiation and its underlying regulatory mechanism.MethodsTo investigate the effects of TNSO on adipocyte differentiation, 3T3-L1 cells were induced to differentiate for 5 days in the presence of 0.75 μL/mL TNSO. Oil Red O staining and an assay for intracellular triglyceride were performed to determine the extent of lipid accumulation in 3T3-L1 cells. To elucidate the underlying mechanism of TNSO, adipogenic gene expression was analyzed using quantitative real-time PCR. Moreover, we monitored TNSO-derived activation of PPARγ and STAT3 with 3T3-L1 reporter cell lines engineered to secrete Gaussia luciferase upon the interaction of a transcription factor to its DNA binding element.ResultsOil Red O staining revealed that TNSO improved the differentiation of 3T3-L1 preadipocytes into mature adipocytes. The mRNA levels of adipogenic genes, including adiponectin, fatty acid synthase (FAS) and adipocyte fatty acid-binding protein (FABP4), were upregulated and intracellular triglyceride levels increased upon TNSO treatment. We also established that adipocyte differentiation was improved by TNSO-derived activation of PPARγ and STAT3.ConclusionsOur results suggest that TNSO improves adipocyte differentiation by regulating the activation of adipogenic transcription factors, indicating that it may serve as a potential treatment strategy for adipocyte dysfunction.

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Increased intracellular sequestration of the insulin-regulated aminopeptidase upon differentiation of 3T3-L1 cells.

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